Hearts are made up of different cell populations, such as cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Atrial and ventricular cardiomyocytes are of special interest in heart research and regenerative medicine. They are naturally located within the mammalian heart and can as well be derived from other cell types, e.g. stem cells, by inductive cues. Several intracellular proteins like muscle proteins and/or transcription factors have been described as being differentially expressed in atrial or ventricular cardiomyocytes; atrial cardiomyocytes are characterized e.g. by gene expression of Myl7, Fgf12, Sln, Gja5, Nppa, Tbx5, ventricular cardiomyocytes are characterized e.g. by gene expression of Hey2, Irx4, Lbh, Myh7.
In mouse development, the four-chambered heart is formed by embryonic day (E) 10.5. The sequence of morphological events coincides with chamber-restricted expression of individual sarcomeric proteins in cardiomyocytes of atria and ventricles. As described by Chuva de Sousa Lopes S M et al. ((2006) Dev Dyn 235(7):1994-2002), mRNA of the atrial intracellular muscle protein, myosin light chain 2a (MLC-2a), is highly expressed in the atria, weakly expressed in the trabeculated and undetectable in the compacted myocardium of the ventricles from E14.5 on. Opposite expression is described for the mRNA of the ventricular intracellular muscle protein (MLC-2v), which was found to be expressed in both ventricles but absent in the atria. After birth, expression of both markers is even more restricted to either atria or ventricles. To date, expression of intracellular proteins enriched either in atrial or ventricular cardiomyocytes could not be correlated to corresponding cell surface markers enabling selective cell enrichment of either atrial or ventricular cardiomyocytes. Therefore, it is currently impossible to purify atrial and ventricular cardiomyocytes from mixed cell populations as well as from mixed cardiomyocyte populations by means of cell surface marker-based cell separation procedures.
The expression patterns of key cardiac genes during in vitro pluripotent stem cell (PSC) differentiation is known to closely reflect their endogenous expression during in vivo cardiogenesis and PSC-derived cardiomyocytes share functional characteristics with embryonic cardiomyocytes (Hescheler et al. (1997) Cardiovasc Res 36:149-162). A major issue is still the heterogeneity of cell populations generated by in vitro differentiation protocols. Besides contaminating non-cardiomyocytes, most differentiation protocols generate a mixture of cardiomyocyte subpopulations, like atrial, ventricular and pacemaker cardiomyocytes. Although differentiation protocols favouring generation of cardiomyocyte subpopulations have been reported, neither generation of pure cardiomyocyte subpopulations nor surface marker-based enrichment of individual subpopulations have been described. Cardiomyocytes are usually identified by antibody-based immunofluorescence analysis, using antibodies against cardiomyocyte-specific transcription factors or sarcomeric proteins or electrophysiological measurements. All known subtype-specific proteins are intracellularly localized and thus prevent isolation of viable cells, because the antibodies are not able to bind intracellular components without destroying cells by permeabilization to allow for antibody penetrance, thereby limiting downstream analysis and preventing use of viable cardiomyocyte subpopulations.
Although general cell surface markers of primary and PSC-derived cardiomyocytes, unable to discriminate between atrial and ventricular cardiomyocytes, have been described and surface marker-based enrichment has been performed e.g. with human PSC-derived cardiomyocytes, methods for separation of primary or PSC-derived cardiomyocytes into subpopulations of atrial and ventricular cardiomyocytes by means of surface marker-based cell purification methods are still missing.
Cell surface marker-independent experimental procedures have been described to enrich cardiomyocytes from mixed cell populations, but are still unable to discriminate between atrial and ventricular cardiomyocytes: 1) physical separation based on size: cardiomyocytes accumulate in a specific layer of a Percoll® gradient. This method is laborious, gives purities of 75-90% as well as very weak cell yield. 2) fluorescent reporter or antibiotic resistance genes that are driven by a cardiomyocyte- or subtype-specific promoters, e.g. myosin light chain (Bizy et al. (2013) Stem Cell Res 11:1335-1347). This yields >90% cardiomyocytes after FACSorting or antibiotic selection, but requires genetic modification of every single cell or mouse line and is neither broadly applicable nor useful for clinical translation. 3) Molecular beacons that emit a fluorescence signal when hybridized to target mRNAs, like cardiac troponin T or myosin light chains (Ban et al. (2013) Circulation 128:1897-1909). This method again requires (genetic) modification of cells by transfection of respective molecular beacons. 4) High abundance of cellular mitochondria is a common characteristic of primary cardiomyocytes. Therefore, intracellular labeling of cardiomyocytes with mitochondrial dyes like MitoTracker® Red has been described as a tool to enrich cardiomyocytes by FACSorting (Hattori et al. (2010) Nat Methods 7:61-66), but turned out to be useful only in primary cardiomyocytes that accumulated high numbers of mitochondria. 5) Metabolic selection of cultured cardiomyocytes by exchange of glucose with lactate in the culture medium has been described as a means to enrich for cardiomyocytes (Tohyama et al. (2013) Cell Stem Cell 12:127-137); major disadvantages of this method are a lengthy selection period (several days) as well as weak recoveries of cardiomyocytes.
None of these methods can be used to selectively enrich for cardiomyocyte subpopulations, i.e. atrial and ventricular cardiomyocytes from mixed cell populations. Besides, several cell surface markers have been identified on primary or PSC-derived cardiomyocytes, but not on atrial or ventricular cardiomyocyte subpopulations and hence are not suitable for the discrimination of atrial and ventricular cardiomyocytes. Most cell surface markers described so far are expressed on cardiomyocytes as well as on non-cardiomyocytes, like CD106 (VCAM-1), CD166 (ALCAM), CD340 (ErbB2) and CD61 (Integrin beta-3).
Stuart Walsh ((2010) Dissertation, Lund University; ISBN 978-91-86443-59-7) identified VCAM-1 (CD106) as cardiomyocyte cell surface marker by flow cytometry analysis of alpha-MHC promoter-eGFP expression in embryonic mouse heart cells. FACS sorting revealed that >97% of CD106+/CD31− sorted cells from embryonic day 10.5-11.5 embryos were also positive for the cardiac muscle protein Troponin T. Sorted cells expressed cardiac specific structural proteins including alpha-MHC, MLC-2a and MLC-2v, indicating that this cell surface marker labels atrial and ventricular cardiomyocytes and does not allow for subtype-specific isolation. Antibodies against VCAM-1 could as well be used to purify a mixture of ventricular-like and pacemaker-like cardiomyocytes derived from human PSCs. (Uosaki et al. (2011) PLoS One 6:e23657). Hirata et al. ((2006) Cells Tissues Organs 184:172-80) identified ALCAM (CD166) as general surface marker for cardiomyocytes in mouse hearts between embryonic day 8.25 and 10.5 by immunofluorescence analysis. Additionally, Rust et al. ((2009) Regen Med 4:225-237) used antibody-based enrichment of ALCAM-positive, human PSC-derived cardiomyocytes. Nevertheless, ALCAM expression cannot be used to discriminate between cardiomyocyte subpopulations.
Although not yet explicitly described for antibody-based cardiomyocyte enrichment, expression of CD340 (ErbB2) and ErbB4 in primary and human/mouse PSC-derived cardiomyocytes of the working myocardium is known (e.g. Pentassuglia and Sawyer (2009) Exp Cell Res 315:627-637). Expression of several integrin family members in cardiomyocytes is described: alpha-1, alpha-3, alpha-5 (CD49e), alpha-6 (CD49f), alpha-7, alpha-9, and alpha-10 as well as beta subunits beta-1, beta-3 (CD61) and beta-5 were found to be expressed in primary or PSC-derived cardiomyocytes. The main integrin heterodimers on the cardiomyocyte surface are alpha-5/beta-1 and alpha v/beta-3 (Ross and Borg (2001) Circ Res 88:1112-1119). Characterization of integrin family member distribution based on in situ hybridization (mRNA) and immunohistochemistry (protein) analysis found high expression of integrin alpha-6 (CD49f) in the atria throughout development. Integrin alpha-6 is absent from the compact layer of the ventricles, but highly expressed in the ventricular trabeculae from E15 onwards. Additionally, integrin alpha-6 expression in the endocardium reaches a peak at E18, including all coronary endothelial cells. Extracardially, alpha-6 was found in endothelium, epithelia, and nervous tissue (Hierck et al. (1996) Dev Dyn 206:100-111). These data clearly indicate a simultaneous expression of integrin alpha-6 in atrial and ventricular cardiomyocytes as well as non-cardiomyocytes during heart development. Single cell analysis as well as a direct correlation of integrin alpha-6 expression with cardiomyocyte subpopulation specific, intracellular proteins expression (e.g. MLC-2a) in the same cell is missing. Additionally, no data are provided on technology development using integrin expression patterns for the development of cell separation strategies for selective enrichment of atrial and/or ventricular cardiomyocytes.
The current inability to enrich for atrial and/or ventricular cardiomyocytes from mixed cell populations containing cardiomyocytes and non-cardiomyocytes or preparations of cardiomyocytes containing mixtures of cardiomyocyte subpopulations by a surface marker-based enrichment method, prevents the use of atrial and/or ventricular cardiomyocytes for downstream applications like:                a) individual drug screening approaches on enriched atrial or ventricular cardiomyocytes        b) cell replacement therapy using enriched cardiomyocyte subpopulations, e.g. ventricular-like cardiomyocytes for transplantation into the ventricle, atrial-like cardiomyocytes for transplantation into the atrium        c) characterization of cardiomyocyte subtype emergence during heart development        d) selective targeting of cardiomyocyte subpopulations for gene therapy and drug delivery applications, using e.g. cardiomyocyte subtype-specific single chain antibodies        
Therefore, there is a need in the art for a method for enrichment, isolation, detection and/or analysis of atrial and ventricular cardiomyocytes.